Variability of allergen-based length polymorphism of Glycine max L. varieties

: Food allergies are an increasingly common phenomenon across all age groups and can be


Introduction
Legumes are essential crops thanks to their nutrition and growing attributes.They represent 27% of global primary crop production.Legumes, also with cereals, are considered an elementary part of nourishment because of the high level of proteins.Unfortunately, they produce a few antinutritional proteins which can act as allergens, digestive enzymes inhibitors, non-proteinogenic amino acids (Smýkal et al., 2015) or lectins.Allergens are a significant group causing immune responses from a mild OAS (Oral Allergy Syndrome) to a severe anaphylactic shock, which can lead to death.In legumes, the most important allergens are profilin, actin-binding protein, and vicilin, protein classified to 7/8S globulin group.Globulins are dominant allergens that the law draws attention to, therefore the soy or peanuts are highlighted in "ingredient part" of food products (Bennetau-Pelissero, 2019).
Globally, Glycine max L. is used to make food, feed or as a part of processing industry (Colletti et al., 2020).Soybean is a great source of proteins comparable with animal products (meat, eggs, or milk caseins), and a soy oil contains relevant quantity of saturated and unsaturated fatty acids, polysaccharides, dietary fiber, phytosterols and saponins (Modgil et al., 2021).Like other legumes, it contains antinutritional substances, especially antinutritional proteins.Soybean is one of eight main foods causing more than 90% food allergies (Verma et al., 2012).Several soy allergens were described: non-specific LTP, defensin, profilin, PR-proteins, vicilin, legumin, 2S albumins or cysteine proteases (Breiteneder a Radauer, 2004).
The aim of this study was an application of DNA marker technique based on isoforms length polymorphism of the soy allergens in soybean varieties with determination of intraspecies variability at the genomic level.

Plant material
Biological material consisted of a soy plants, grown in the field conditions in May 2021 (average temperature: 20 °C, day-length: 15h, number of rainy days: 11) and in vitro conditions in growth chamber (parameters simulated by yield conditions) in AgroBioTech Research Centre in Nitra (Slovakia).The choice of varieties was very random to increase possibility of a genome variability.The only condition was an equal representation of regional, high-yielding and rare varieties (Table 1).

PBAP and VBAP analysis
Profilin variability was examined by two set of primers -specific and degenerated.Specific primers were designed to fruit profilins (Klongová, Kováčik et al., 2021), degenerate primers were designed on the base of conservative parts of profilin found in sequences of Rosaceae (taxid:3745) in NCBI database.(Table 2).
Profiles were divided into 3 groups (Figure 1), the most abundant was the second including 'McCall', 'Accord', 'Gaillard', 'Mario', 'Rigel', 'Quito', 'Emerson', 'Bristol', 'Belmont', 'Crystal', whereas 'Emerson' and 'Bristol' were the most similar (sharing 81,25 % identity) and the most dissimilar was 'Mario' (only 25 % similarity).The most different profile of whole material collection produced variety 'Mario', on the other hand, 100% identical profiles shared 'Danica' and 'Schlaming'.Profilin isoforms were identified in all varieties by specific primers, their profiles showed high level of intraspecies polymorphism.Degenerated primers for profilin isoforms were not able to catch area of interest.PCR products were amplified only in varieties 'Dorota' and 'Bristol' with identical profiles of 2 alleles -800 bp and 1000 bp.
The expression levels of allergens can vary due to various factors, such as plant cultivars (Marzano et al., 2020), growth and storage conditions and ripening stages.Proteomics can also yield much information about the expression levels of plant allergens in various environmental conditions (Nakamura and Teshima, 2013).One of the factors that affect allergen expression levels is the genetic background of plant cultivars (Marzano et al., 2020).In future, polymorphism of profilin genes could uncover one of the reasons for the variability of observed expression or immune reactions to soy.In addition to environmental factors, point to the genetic background that affects the expression level of pathogen related proteins, which also include allergens from the profilin family.

VBAP of analysed G. max varieties
VBAP technique was used to identify the presence of vicilin and to uncover possible intraspecies polymorphism in a captured genome region.The primers were able to amplify 36 alleles in 17 loci with following lengths: 106 bp, 126 bp, 151 bp, 179 bp, 255 bp, 285 bp, 300 bp, 327 bp, 402 bp, 500 bp, 560 bp, 596 bp, 634 bp, 714 bp, 762 bp, 813 bp a 929 bp.Primers were not able to catch vicilin fragments into genomes of 'Maple Ridge', 'OAC Scorpio', 'Nattong-K87', 'AC Glengarry' and 'Gaillard' (not included in dendrogram).Vicilin, as a storage protein, is essential for the plant, so it is a small possibility of its gene to absent in soy genome.However, it is most likely that the inability of the VBAP technique to catch the gene points to a high level of gene variability.To the date, 5 isoforms were described by Allergen Nomenclature Database, database UniProt showed 8 β-conglycinin subunits more than 90% similarity in amino acid sequences.
Similar as the BPAP results, varieties were divided into 3 bigger groups (Figure 2), but one of them included only 2 identical profiles of 'Schlaming' and 'Krajina' which were dissimilar to the rest profiles.On the other hand, absolute dissimilar profiles created 'Maverick' and 'Danica'.The most similar profiles (excluded identical) had varieties 'Ugo' and 'Belmont' sharing 44 % of alleles.

Conclusion
Soybean is a good resource of important substances, but it also contains antinutritional chemicals, such as allergens.The most significant are profilin and vicilin.To understand different levels of allergy reactions to both, it is relevant to identify their isoforms and to describe the differences among them.Therefor PBAP and VBAP techniques were used to 30 varieties of G. max, PBAP created 29 polymorphic profiles, VBAP made 25, of which 2 were unique.VBAP technique was not able to amplify selected sequences in genomes of 5 varieties ('Maple Ridge', 'OAC Scorpio', 'Nattong-K87', 'AC Glengarry' and 'Gaillard') which are proper to next research.

Table 1 .
List of used Glycine max L. varieties used.
(Thermo Scientific).Its functionality has been verified by ITS technique at a 1:9 dilution.